Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that is the causative agent of human acquired immunodeficiency syndrome (AIDS). In retroviruses, tRNA molecules are recruited as primers to initiate reverse transcription. In the case of HIV-1, a specific host cell tRNA, human tRNA LyS'3, serves as the replication primer. The 3' 18 nucleotides of this tRNA are unwound and annealed to the primer binding site (PBS) in a process that is facilitated by the HIV nucleocapsid protein (NC). Previous studies primarily using heat-annealed complexes, have suggested that a specific ruination complex forms between the tRNA primer and the genome that involves extensive interactions outside the PBS region. This proposal will explore this hypothesis further using NC-annealed complexes. RNA structural changes that occur upon HIV-1 NC binding to the HIV-1 genome and to the tRNA-genome initiation complex will be elucidated. We will also probe the functional significance of the interactions outside the PBS that are formed upon tRNA-Lys,3 annealing by HIV- 1 NC. The goals will be accomplished by employing nucleotide analog interference mapping (NAIM) experiments, metal ion cleavage assays, fluorescence resonance energy transfer (FRET) studies, and crosslinking experiments. Information gained from the proposed research will provide valuable insights into the molecular interactions that contribute to the specificity of NC-tRNA primer and NC-tRNA primer-HIV genome interactions. Additionally, this information may be useful in the design of new therapeutic agents against AIDS and retroviral-derived cancers.